The invention relates to novel compositions for the preparation of mature dendritic cells as well as to methods for in vitro maturation of immature dendritic cells and to therapeutic uses of the dendritic cells obtainable by the method of the invention.
Dendritic cells (DCs) have a high potential as adjuvants in the induction of tumor-specific killer and helper cells in the patient (Schuler G, Schuler-Thurner B, Steinman R M. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol. 2003 April; 15(2): 138-47. Review. Banchereau J, Palucka A K. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol. 2005 April; 5(4):296-306. Review., Salcedo M, Bercovici N, Taylor, Vereecken P, Massicard S, Duriau D, Vernel-Pauillac F, Boyer A, Baron-Bodo V, Mallard E, Bartholeyns J, Goxe B, Latour N, Leroy S, Prigent D, Martiat P, Sales F, Laporte M, Bruyns C, Romet-Lemonne J L, Abastado J P, Lehmann F, Velu T. Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate. Cancer Immunol Immunother. 2005 September 27:1-11 [Epub ahead of print]).
For this purpose, mature dendritic cells which have been maturated in vitro from immature dendritic cells derived from the patient, are loaded with tumor-specific antigens and reinjected into the body, preferably next to or in the lymph nodes. Within lymph nodes dendritic cells interact with-naive T cells resulting in active signal transduction during the so called immunological synapse and subsequent proliferation of effector T cells, which, in turn mediate anti tumor responses like cytotoxicity (cytotoxic T lymphocytes=CTLs), activation of macrophages and delayed type hypersensitivity reactions. DCs regulate CD4 positive T helper (h) cell polarizations. Th1 cells, for example, support CTLs by secretion certain cytokine patterns (e.g. Interferon gamma and IL-2, TNF-beta). On the other hand, Th2 cells induce antibodies as well as eosinophiles and degranulation of mast cells by IL-4, IL-5, IL-10 and IL-13 (Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat. Immunol. 2000 October; 1(4):311-6, O'Gara, A: Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 1998, 8: 275-283, Rengarajan J, Szabo S J, Glimcher L H. Transcriptional regulation of Th1/Th2 polarization. Immunol Today. 2000 October; 21(10):479-83. Review).
For the therapy with dendritic cells, it is essential that a sufficient number of major DCs is available. Since, in the patient, only 0.2% of the white blood cells are dendritic cells, it is necessary to have an efficient method for the in vitro production of mature dendritic cells.
In the art, various methods have been proposed for the preparation of mature dendritic cells starting from peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells (Jonuleit H, Kuhn U, Muller G, Steinbrink K, Paragnik L, Schmitt E, Knop J, Enk A H.: Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur J. Immunol. 1997 December; 27(12):3135-42), Mosca P J, Hobeika A C, Clay T M, Nair S K, Thomas E K, Morse M A, Lyerly H K. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood. 2000 Nov. 15; 96(10):3499-504, Mailliard R B, Wankowicz-Kalinska A, Cai Q, Wesa A, Hilkens C M, Kapsenberg M L, Kirkwood J M, Storkus W J, Kalinski P.: alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 2004 Sep. 1; 64(17):5934-7, Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A.: Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat. Immunol. 2005 August; 6(8):769-76. Epub 2005 Jul. 3, 2005 August; 6(8):769-76.2005, Gautier G, Humbert M, Deauvieau F, Scuiller M, Hiscott J, Bates E E, Trinchieri G, Caux C, Garrone P.: A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J Exp Med. 2005 May 2; 201(9):1435-46, 2005).
It is accepted that the cultivation of peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells with GM-CSF and either IL-4 or IL-13 results in the production of immature dendritic cells in vitro (Ahn J S, Agrawal B. IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells. Int Immunol. 2005 October; 17(10):1337-46. Epub 2005 Sep. 2.) However, to date, there is no satisfying method available for the maturation of the immature dendritic cells. Jonuleit H. et al. (1997, supra) describe such a maturation process using a composition comprising TNF-α, IL-1, IL-6 and prostaglandin E2 (PG) (the so-called Jonuleit cocktail). Dendritic cells produced by incubation of immature dendritic cells with this composition show the surface markers for mature dendritic cells and can be well harvested. However, these cells fail to produce biological active IL-12p70, which is the most important factor for the induction of Th1 cells in the lymph nodes.
Mailliard, R. et al. describe a composition comprising TNF-α, IL-1, interferon α, interferon γ and polyI:C (Mailliard, R. et al., 2004, supra). In contrast to the above Jonuleit cocktail, incubation of immature dendritic cells with this so called Kalinski cocktail results in mature dendritic cells (as demonstrated by the respective surface markers), which produce IL-12p70. However, these cells are very adherent to the bottom of the culture flasks and are, therefore, nearly impossible to harvest. It is, therefore, very difficult, if not impossible, to obtain sufficient mature dendritic cells for the vaccination therapy with this method.
WO 00/47719 describes a compound (R848) which is proposed for the preparation of mature dendritic cells. In the experiments described in this application, immature dendritic cells are stimulated with R848 only. However, R848 as a single maturation substance is not able to provide all characteristics suitable for clinical dendritic cells. All experiments have been carried out with FCS (fetal calf serum) and, therefore, not applicable under GMP (good manufacturing process) conditions because fetal calf serum-free conditions are crucial for a GMP process.
Therefore, there is still a need for improved methods for the preparation of mature dendritic cells out of immature dendritic cells.
The invention provides a method for in vitro maturation of at least one immature dendritic cell, comprising stimulating said immature dendritic cell with TNF-α, IL-1β, IFNγ, a TLR7/8 agonist and prostaglandin E2 (PG).
The present invention is based on the surprising finding that the combination of TNF-α, IL-1β, IFNγ, a TLR7/8 agonist and prostaglandin E2 (PG) is especially suitable for promoting the in vitro maturation of dendritic cells. Especially, and as demonstrated in the Example, the mature dendritic cells obtained by using said combination surprisingly express IL-12p70 in considerable amounts and are surprisingly easy to harvest, which allows for obtaining mature dendritic cells in considerable amounts. Such mature dendritic cell populations could not be produced with the cocktails known in the art, and especially not with the Jonuleit cocktail or the Kalinski cocktail, as explained above.
Individual techniques for the preparation of mature dendritic cells, e.g. starting from human peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells, and from immature DCs themselves, which have been directly isolated from the blood, are known in the art (Berger T G, Strasser E, Smith R, Carste C, Schuler-Thurner B, Kaempgen E, Schuler G. Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells. J Immunol Methods. 2005 March; 298(1-2):61-72. Erratum in: J Immunol Methods. 2005 August; 303(1-2):152, Strasser E F, Berger T G, Weisbach V, Zimmermann R, Ringwald J, Schuler-Thurner B, Zingsem J, Eckstein R. Comparison of two apheresis systems for the collection of CD14+ cells intended to be used in dendritic cell culture. Transfusion. 2003 September; 43(9):1309-16. Erratum in: Transfusion. 2003 October; 43(10):1502, Campbell J D, Piechaczek C, Winkels G, Schwamborn E, Micheli D, Hennemann S, Schmitz J. Isolation and generation of clinical-grade dendritic cells using the CliniMACS system. Methods Mol. Med. 2005; 109:55-70, Dubsky P, Ueno H, Piqueras B, Connolly J, Banchereau J, Palucka A K. Human dendritic cell subsets for vaccination. J Clin Immunol. 2005 November; 25(6):551-72).
Therefore, the basic techniques such as incubation periods, media used, etc., for producing mature dendritic cells out of immature dendritic cells, are known in the art. The present invention relates to a novel combination of factors to be used in the context of these prior art techniques. The method of the present invention can, therefore, be easily practiced by the person skilled in the art, simply by performing prior art methods, but using the above identified combination of factors during the incubation of immature dendritic cells in order to obtain mature dendritic cells.
Furthermore, since each of the individual components has already been individually used in the art, the person skilled in the art can easily determine in which concentration each factor should be used. Additionally, the skilled person would be able to adapt the individual concentration of a given factor depending on compositions of the cell culture medium especially growth factors and serum components.
As a general guidance, TNF-α and IL-1β might be used at concentrations from 1 ng/ml to 50 ng/ml, more preferably from 5 ng/ml to 40 ng/ml, and even more preferably at 10 ng/ml. PG might be used at concentrations from 50 ng/ml to 5000 ng/ml, preferably from 50 ng/ml to 1000 ng/ml, even more preferably from 50 ng/ml to 500 ng/ml or at 100 ng/ml or 250 ng/ml. IFNγ might be used at a concentration between 500 U/ml and 10000 U/ml, preferably between 1000 and 5000 U/ml, and more preferably either at 1000 or 5000 U/ml. Finally, the TLR7/8 agonist, preferably R848, might be used at a concentration between 0.2 and 5 g/ml, preferably 0.5 μg/ml to 2 μg/ml, more preferably 1 μg/ml.
According to the invention, immature dendritic cells are cultivated with the above combination of factors. This can be performed by adding the factors to the culture medium. Alternatively, the culture medium in which the immature dendritic cells have been grown is replaced by a medium already containing the factors. In a further preferred embodiment, the substances mentioned above are part of a composition added to the culture medium of said immature dendritic cell.
Said culture medium may be of any suitable kind, i.e. it may contain human serum or not, may be supplemented with or without any other animal supplements, like proteins, amino acids, or antibiotics. In a preferred embodiment, the medium is produced and used under GMP conditions.
After the maturation period is completed, DCs may be harvested by up and down pipetting, shaking (by hand or mechanically) and rinsing with salt solution, medium components (e.g. RPMI) or complete medium without cytokines. Cells may be collected, centrifuged and cytokines may be washed out by at least one more resuspension of pelleted DCs.
The immature dendritic cells may further be treated with a TLR3 ligand, preferably polyI:C, e.g. at a concentration of between 10 and 50 ng/ml, preferably 20 ng/ml. TLR3 ligand may be added separately to the cells or may be part of the composition comprising also the other factors.
In a preferred embodiment of the invention, said TLR7/8 agonist is an imidazoquinilone type immune response modifying compound, more preferably 4-amino-2-ethoxymethyl-α,α-dimethyl-1H-imidazol[4,5-c]quinoline-1-ethanol (R848). The production of such compounds is described in detail in WO 00/47719. However, also other TLR7/8 agonists as imiquimod (R837), guanine analog loxoribine, TLR8 agonists as single-stranded RNAs which bind to TLR7/8, e.g. ss polyU and ss RNA40 or combinations of TLR7/8 agonists may be used.
In a further preferred embodiment, the immature dendritic cell used as the starting material of the method of the invention is a monocyte derived immature dendritic cell. Preferably, monocytic progenitors obtained from peripheral blood or leukapheresis and enriched by density gradient centrifugation, elutriation or simply plastic adherence techniques are used.
Alternatively, it is also possible to obtain monocytic progenitor cells from CD34 positive progenitor cells by in vitro differentiation to CD14 positive cells, e.g. with FLT3L, SCF, TPO, 11-3 and/or IL-6.
Preferably, said immature dendritic cell is obtained by incubating human peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells with GM-CSF and IL-4 or IL-13. As already discussed above, corresponding methods are known in the art. Furthermore, such methods are described in the Example.
Any medium suitable for physiological conditioning of mammalian cells e.g. containing standard amino acids, growth factors, carbon source, buffer system, or certain salts may be used. Cell culture may be performed at 37° C. according to medium composition at certain CO2 concentrations.
Furthermore, the immature DC may be obtained directly from peripheral blood e.g. via leukapheresis.
In an especially preferred embodiment, the immature dendritic cell is of human origin, although situations, e.g. scientific research or veterinary medicine applications, may be feasible where immature dendritic cells of mammalian origin may be used.
Consequently, in a further preferred embodiment, the method of the invention comprises the following steps:                a) preparing mononuclear cells from peripheral blood,        b) incubating the mononuclear cells of step a) with GM-CSF and IL-4 or IL-13,        c) incubating the cells obtained in step b) with a cocktail comprising TNFα, IL-1β, IFNγ, a TLR7/8 agonist, prostaglandin E2 (PG), and, optionally, a TLR3 agonist, preferably polyI:C, and        d) harvesting the mature dendritic cell or cells.        
In step a), the mononuclear cells may be obtained by leukapheresis from peripheral blood. Furthermore, mononuclear cells may be isolated by magnetic or FACS sorting, elutriation or plastic adherence or density gradient centrifugation (e.g. metricamide)
Preferably, the incubation in step b) takes 1 to 9, preferably 2 to 9, more preferably 2 to 6 days. However, it is also feasible to spare steps a) or b) if using freshly isolated immature DCs from peripheral blood/leukapheresis. Furthermore, it is possible that step b) lasts only hours and may be performed in combination with step c).
The incubation in step c) may take 12 hours to 72 hours, preferably 24 hours or 20 hours.
As already discussed above, the skilled person will be able to adapt these incubation periods, if necessary.
The incubation of the immature dendritic cells and the harvesting have already been described above.
In a further preferred embodiment of the invention, the mature dendritic cell or cells is/are further loaded in vitro with one or more antigens. The loading of the mature cells with said antigens is described below in more detail.
Preferably, said antigen or antigens are supposed to trigger the effector T cell maturation within the lymph nodes.
More preferably, and as also described below, said loading is performed by incubating the mature dendritc cell or cells with at least one peptide of said antigen or by transfecting the dendritic cell or cells with antigen encoding RNA or DNA.
The invention further relates to a mature dendritic cell or population of mature dendritic cells, obtainable by the method of the invention. As discussed above, the mature dendritic cells obtained by the method of the invention produce considerable amounts of IL-12p70 and are easy to harvest. These combined effects were not observed with the Jonuleit or Kalinski cocktail in the experiments presented herein (see Example).
As demonstrated in the Examples and especially in FIGS. 7 and 8, the population of mature dendritic cells of the invention is capable of stimulating interferon-gamma production both of natural killer cells (FIG. (8) as well as of peptide-specific T cells (FIG. 7). Consequently, the dendritic cells obtainable by the method of the invention are especially suitable in the context of activating T cells in vivo, in order to treat diseases where such activation of T cells is necessary. Consequently, in a further aspect, the present invention also relates to a pharmaceutical composition comprising the mature dendritic cell or the mature dendritic cells. Furthermore, the invention also relates to the use of the mature dendritic cell or of the population of mature dendritic cells of the invention for the preparation of a medicament for the treatment of a disease selected from the group consisting of tumorigenic diseases, and infectious diseases (e.g. provoked by viruses, bacteria, intracellular bacteria or fungi).
In a preferred embodiment, said dendritic cells are obtainable by a method of the invention wherein the cells are incubated also with poly I:C. As indicated above, such dendritic cells are especially capable of stimulating NK cells and are as potent as cells incubated according to the invention without poly I:C in stimulating peptide-specific T cells.
Preferably, for the treatment of the above diseases, the dendritic cells are loaded in vitro with antigens supposed to trigger the effector T cell maturation within the lymph nodes. Such techniques are known in the art (Dieckmann D, Schultz E S, Ring B, Chames P, Held G, Hoogenboom H R, Schuler G. Optimizing the exogenous antigen loading of monocyte-derived dendritic cells. Int Immunol. 2005 May; 17(5):621-35. Epub 2005 Apr. 11, Kikuchi T, Akasaki Y, Abe T, Fukuda T, Saotome H, Ryan J L, Kufe D W, Ohno T. Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin 12. J Immunother. 2004 November-December; 27(6):452-9, Gong J, Koido S, Kato Y, Tanaka Y, Chen D, Jonas A, Galinsky I, DeAngelo D, Avigan D, Kufe D, Stone R. Induction of anti-leukemic cytotoxic T lymphocytes by fusion of patient-derived dendritic cells with autologous myeloblasts. Leuk Res. 2004 December; 28(12):1303-12, Grunebach F, Kayser K, Weck M M, Muller M R, Appel S, Brossart P. Cotransfection of dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes. Cancer Gene Ther. 2005 September; 12(9):749-56, Kyte J A, Kvalheim G, Aamdal S, Saeboe-Larssen S, Gaudernack G. Preclinical full-scale evaluation of dendritic cells transfected with autologous tumor-mRNA for melanoma vaccination. Cancer Gene Ther. 2005 June; 12(6):579-91, Navabi H, Croston D, Hobot J, Clayton A, Zitvogel L, Jasani B, Bailey-Wood R, Wilson K, Tabi Z, Mason M D, Adams M. Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial. Blood Cells Mol. Dis. 2005 September-October; 35(2):149-52, Escudier B, Dorval T, Chaput N, Andre F, Caby M P, Novault S, Flament C, Leboulaire C, Borg C, Amigorena S, Boccaccio C, Bonnerot C, Dhellin O, Movassagh M, Piperno S, Robert C, Serra V, Valente N, Le Pecq J B, Spatz A, Lantz O, Tursz T, Angevin E, Zitvogel L. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J Transl Med. 2005 Mar. 2; 3(1): 10, Kawamura K, Kadowaki N, Suzuki R, Udagawa S, Kasaoka S, Utoguchi N, Kitawaki T, Sugimoto N, Okada N, Maruyama K, Uchiyama T. Dendritic cells that endocytosed antigen-containing IgG-liposomes elicit effective antitumor immunity. J Immunother. 2006 March-April; 29(2):165-74, Griffioen M, Borghi M, Schrier P I, Osanto S, Schadendorf D. Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates. Cancer Immunol Immunother. 2004 August; 53(8):715-22. Epub 2004 Mar. 3, Su Z, Dannull J, Yang B K, Dahm P, Coleman D, Yancey D, Sichi S, Niedzwiecki D, Boczkowski D, Gilboa E, Vieweg J. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer. J. Immunol. 2005 Mar. 15; 174(6):3798-807).
Loading of dendritic cells with respective antigens could be by competitive displacement of peptides within solutions from the MHC binding groove, or for more complex antigens, like proteins and original tumor lysates or lysates of tumor cell lines, through phagocytosis of immature DCs and proper processing. Transfection methods (lipofection, electroporation or simply incubation of naked nucleic acids) are also feasible and introduce nucleic acids, such as antigen encoding plasmids, RNA of them or DNA and especially RNA from original tumors or tumor cell lines into the DCs. There might also be other antigenic combinations with original MHC molecules conceivable such as membrane fragments or exosomes to use as antigen sources of any kind.
As indicated above, the dendritic cells can be administered directly to the organism to produce T cells active against a selected, e.g. cancerous cell type. Administration of these cells, often with pharmaceutically acceptable carriers, is by any of the routes normally used for introducing a cell into ultimate contact with a mammal's blood or tissue cells.
Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes (preferably intradermal or subcutaneous), and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous or intraperitoneal administration are the preferred method of administration for dendritic cells of the invention.
The dose of the dendritic cells administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit growth of cancer cells, or to inhibit infection. Thus, cells are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” The dose will be determined by the activity of dendritic cell produced and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular cell in a particular patient. In determining the effective amount of the cell to be administered in the treatment or prophylaxis of diseases such as cancer (e.g., metastatic melanoma, prostate cancer, etc.), the physician needs to evaluate circulating plasma levels, CTL toxicity, progression of the disease, and the induction of immune response against any introduced cell type.
Prior to infusion, blood samples are obtained and saved for analysis. Generally at least about 104 to 106 and typically, between 108 and 1010 cells are infused intravenously or intraperitoneally into a 70 kg patient over roughly 60-120 minutes. Preferably, cell numbers of at least 107/vaccination point are used. The injections may be e.g. 4 times repeated in a 2 weeks interval and should be given preferably near lymph nodes by intradermal or subcutaneous injections. Booster injections may be performed after a 4 weeks pause. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for analysis. Cell reinfusion are repeated roughly every month for a total of 10-12 treatments in a one year period. After the first treatment, infusions can be performed on a outpatient basis at the discretion of the clinician. If the reinfusion is given as an outpatient, the participant is monitored for at least 4 hours following the therapy.
For administration, cells of the present invention can be administered at a rate determined by the LD-50 (or other measure of toxicity) of the cell type, and the side-effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. The cells of this invention can supplement other treatments for a condition by known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic response modifiers. Similarly, biological response modifiers are optionally added for treatment by the dendritic cells. For example, the cells are optionally administered with an adjuvant, a cytokine such as GM-CSF, IL-12, or IL-2, or with KLH.
As indicated above, the invention also relates to the combined use of TNF-α, IL-1β, IFNγ, a TLR7/8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably polyI:C for the preparation of at least one mature dendritic cell. Furthermore, the invention also relates to a composition comprising TNF-α, IL-1β, IFNγ, a TLR7/8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably polyI:C. As indicated above, in both cases, preferably said TLR7/8 agonist is an imidazoquinilone type immune response modifying compound, preferably 4-amino-2-ethoxymethyl-α,α-dimethyl-1H-imidazol[4,5-c]quinoline-1-ethanol (R848).
The invention will be further described by the following figures and examples, which are not intended to limit the scope of protection as defined in the claims.